Every year 700,000 Americans are afflicted by stroke and 1.5 million Americans currently suffer from Parkinson's Disease. These are just two of several debilitating pathologies involving glutamate "excitotoxicity", a mechanism of neuronal injury caused by excessive release of the neurotransmitter glutamate and consequent overactivation of calcium-permeable NMDA-type glutamate receptors. Drugs that block these receptors have been largely unsuccessful in clinical trials and have serious side effects, underscoring the need for alternative targets of intervention. Two prime events that occur downstream of NMDA receptor activation are dysfunction of mitochondria, the pivotal calcium buffering and energy generating ("bioenergetic") organelle of the cell, and activation of calcium-dependent calpain proteases. Although calpain inhibitors have shown early promise in small animal disease models, very little is known about the targets of calpain proteases and whether calpains contribute to mitochondrial dysfunction. To achieve the eventual goal of designing rational therapeutics for neurodisease, it is critical to understand when and how these proteases function in the neurodegenerative process. This study will test the central hypothesis that calpains play a causative role in the mitochondrial dysfunction and deregulation of calcium homeostasis that are characteristic of neuronal excitotoxicity. The experiments in aim 1 of this study will develop a sensitive and specific fluorescent indicator for live-cell imaging of intracellular calpain activity. Genetic deletion and RNA interference approaches will rigorously establish the specificity of this novel technique. The experiments in aim 2 of the study will assess the time course, extent, and causal role of calpain activation in glutamate-challenged neurons with respect to changes in intracellular calcium and alterations in mitochondrial function. Two new technologies will be developed that will broadly advance the arena of biomedical research in understanding and treating the pathological consequences of neurodisease. They will allow investigators to: 1. Kinetically measure the activity of destructive proteases in intact neurons in conjunction with key physiological parameters; and 2. Reliably distinguish changes in mitochondria and plasma membrane potentials in injured neurons. This study will provide a strong foundation and powerful tools for the detailed investigation of cellular mechanisms underlying neurodegeneration.